Ling-Ling Chen

The draft genome of sweet orange (Citrus sinensis)

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

The genome sequence of Sea-Island cotton (Gossypium barbadense) provides insights into the allopolyploidization and development of superior spinnable fibres

Gossypium hirsutum contributes the most production of cotton fibre, but G. barbadense is valued for its better comprehensive resistance and superior fibre properties. However, the allotetraploid genome of G. barbadense has not been comprehensively analysed. Here we present a high-quality assembly of the 2.57 gigabase genome of G. barbadense, including 80,876 protein-coding genes. The double-sized genome of the A (or At) (1.50 Gb) against D (or Dt) (853u2009Mb) primarily resulted from the expansion of Gypsy elements, including Peabody and Retrosat2 subclades in the Del clade, and the Athila subclade in the Athila/Tat clade. Substantial gene expansion and contraction were observed and rich homoeologous gene pairs with biased expression patterns were identified, suggesting abundant gene sub-functionalization occurred by allopolyploidization. More specifically, the CesA gene family has adapted differentially temporal expression patterns, suggesting an integrated regulatory mechanism of CesA genes from At and Dt subgenomes for the primary and secondary cellulose biosynthesis of cotton fibre in a “relay race”-like fashion. We anticipate that the G. barbadense genome sequence will advance our understanding the mechanism of genome polyploidization and underpin genome-wide comparison research in this genus.

The Differential Transcription Network between Embryo and Endosperm in the Early Developing Maize Seed

Transcriptomic analysis of maize seed soon after pollination aids understanding of how maize embryo and endosperm are differentially regulated in the early development stage. Transcriptome analysis of early-developing maize (Zea mays) seed was conducted using Illumina sequencing. We mapped 11,074,508 and 11,495,788 paired-end reads from endosperm and embryo, respectively, at 9 d after pollination to define gene structure and alternative splicing events as well as transcriptional regulators of gene expression to quantify transcript abundance in both embryo and endosperm. We identified a large number of novel transcribed regions that did not fall within maize annotated regions, and many of the novel transcribed regions were tissue-specifically expressed. We found that 50.7% (8,556 of 16,878) of multiexonic genes were alternatively spliced, and some transcript isoforms were specifically expressed either in endosperm or in embryo. In addition, a total of 46 trans-splicing events, with nine intrachromosomal events and 37 interchromosomal events, were found in our data set. Many metabolic activities were specifically assigned to endosperm and embryo, such as starch biosynthesis in endosperm and lipid biosynthesis in embryo. Finally, a number of transcription factors and imprinting genes were found to be specifically expressed in embryo or endosperm. This data set will aid in understanding how embryo/endosperm development in maize is differentially regulated.

PlantNATsDB: a comprehensive database of plant natural antisense transcripts

Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a ‘Gene Set Analysis’ module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/.

RiceVarMap: a comprehensive database of rice genomic variations

Rice Variation Map (RiceVarMap, http:/ricevarmap.ncpgr.cn) is a database of rice genomic variations. The database provides comprehensive information of 6 551 358 single nucleotide polymorphisms (SNPs) and 1 214 627 insertions/deletions (INDELs) identified from sequencing data of 1479 rice accessions. The SNP genotypes of all accessions were imputed and evaluated, resulting in an overall missing data rate of 0.42% and an estimated accuracy greater than 99%. The SNP/INDEL genotypes of all accessions are available for online query and download. Users can search SNPs/INDELs by identifiers of the SNPs/INDELs, genomic regions, gene identifiers and keywords of gene annotation. Allele frequencies within various subpopulations and the effects of the variation that may alter the protein sequence of a gene are also listed for each SNP/INDEL. The database also provides geographical details and phenotype images for various rice accessions. In particular, the database provides tools to construct haplotype networks and design PCR-primers by taking into account surrounding known genomic variations. These data and tools are highly useful for exploring genetic variations and evolution studies of rice and other species.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63

Significance Indica rice accounts for >70% of total rice production worldwide, is genetically highly diverse, and can be divided into two major varietal groups independently bred and widely cultivated in China and Southeast Asia. Here, we generated high-quality genome sequences for two elite rice varieties, Zhenshan 97 and Minghui 63, representing the two groups of indica rice and the parents of a leading rice hybrid. Comparative analyses uncovered extensive structural differences between the two genomes and complementarity in their hybrid transcriptome. These findings have general implications for understanding intraspecific variations of organisms with complex genomes. The availability of the two genomes will serve as a foundation for future genome-based explorations in rice toward both basic and applied goals. Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica. Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059–2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

Plant siRNAs from introns mediate DNA methylation of host genes

Small RNAs (sRNAs), largely known as microRNAs (miRNAs) and short interfering RNAs (siRNAs), emerged as the critical components of genetic and epigenetic regulation in eukaryotic genomes. In animals, a sizable portion of miRNAs reside within the introns of protein-coding genes, designated as mirtron genes. Recently, high-throughput sequencing (HTS) revealed a huge amount of sRNAs that derived from introns in plants, such as the monocot rice (Oryza sativa). However, the biogenesis and the biological functions of this kind of sRNAs remain elusive. Here, we performed a genome-scale survey of intron-derived sRNAs in rice based on HTS data. Several introns were found to have great potential to form internal hairpin structures, and the short hairpins could generate miRNAs while the larger ones could produce siRNAs. Furthermore, 22 introns, termed sirtrons, were identified from the rice protein-coding genes. The single-stranded sirtrons produced a diverse set of siRNAs from long hairpin structures. These sirtron-derived siRNAs are dominantly 21 nt, 22 nt, and 24 nt in length, whose production relied on DCL4, DCL2, and DCL3, respectively. We also observed a strong tendency for the sirtron-derived siRNAs to be coexpressed with their host genes. Finally, the 24-nt siRNAs incorporated with Argonaute 4 (AGO4) could direct DNA methylation on their host genes. In this regard, homeostatic self-regulation between intron-derived siRNAs and their host genes was proposed.

Genome-wide comparison of microRNAs and their targeted transcripts among leaf, flower and fruit of sweet orange.

BackgroundIn plants, microRNAs (miRNAs) regulate gene expression mainly at the post-transcriptional level. Previous studies have demonstrated that miRNA-mediated gene silencing pathways play vital roles in plant development. Here, we used a high-throughput sequencing approach to characterize the miRNAs and their targeted transcripts in the leaf, flower and fruit of sweet orange.ResultsA total of 183 known miRNAs and 38 novel miRNAs were identified. An in-house script was used to identify all potential secondary siRNAs derived from miRNA-targeted transcripts using sRNA and degradome sequencing data. Genome mapping revealed that these miRNAs were evenly distributed across the genome with several small clusters, and 69 pre-miRNAs were co-localized with simple sequence repeats (SSRs). Noticeably, the loop size of pre-miR396c was influenced by the repeat number of CUU unit. The expression pattern of miRNAs among different tissues and developmental stages were further investigated by both qRT-PCR and RNA gel blotting. Interestingly, Csi-miR164 was highly expressed in fruit ripening stage, and was validated to target a NAC transcription factor. This study depicts a global picture of miRNAs and their target genes in the genome of sweet orange, and focused on the comparison among leaf, flower and fruit tissues.ConclusionsThis study provides a global view of miRNAs and their target genes in different tissue of sweet orange, and focused on the identification of miRNA involved in the regulation of fruit ripening. The results of this study lay a foundation for unraveling key regulators of orange fruit development and ripening on post-transcriptional level.

Insights into the increasing virulence of the swine-origin pandemic H1N1/2009 influenza virus

Pandemic H1N1/2009 viruses have been stabilized in swine herds, and some strains display higher pathogenicity than the human-origin isolates. In this study, high-throughput RNA sequencing (RNA-seq) is applied to explore the systemic transcriptome responses of the mouse lungs infected by swine (Jia6/10) and human (LN/09) H1N1/2009 viruses. The transcriptome data show that Jia6/10 activates stronger virus-sensing signals, such as the toll-like receptor, RIG-I like receptor and NOD-like receptor signalings, as well as a stronger NF-κB and JAK-STAT singals, which play significant roles in inducing innate immunity. Most cytokines and interferon-stimulated genes show higher expression lever in Jia/06 infected groups. Meanwhile, virus Jia6/10 activates stronger production of reactive oxygen species, which might further promote higher mutation rate of the virus genome. Collectively, our data reveal that the swine-origin pandemic H1N1/2009 virus elicits a stronger innate immune reaction and pro-oxidation stimulation, which might relate closely to the increasing pathogenicity.

Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction

The emergence of apomixis—the transition from sexual to asexual reproduction—is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.